C Cao1, C Shi, P Li, Y Tong, Q Ma. 1. Molecular Genetics Center, Institute of Biotechnology, Academy of Military Medical Sciences, 20 Dongdajie, Fengtai District, Beijing 100071, People's Republic of China.
Abstract
BACKGROUND: Hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis. Detection of circulating antibodies against HCV by enzyme-linked immunosorbent assay (ELISA) has provided the main approach for the diagnosis of HCV infection. Most ELISA kits use a mixture of core, NS3, NS4 and NS5 antigen as capture antigens and enzyme-labeled goat anti-human IgG as conjugate. OBJECTIVES: To establish an ELISA system based on the antigen-capturing principle, using a recombinant chimeric polyprotein containing four HCV antigenic components as antigen. STUDY DESIGN: HCV antigens were expressed in Escherichia coli as chimeric polyprotein either in inclusion bodies or in soluble form. Protein expressed in inclusion bodies was used as solid-phase antigen, and the antigen expressed in a soluble form was used as enzyme conjugate after being labeled with horseradish peroxidase (HRP). RESULTS: Genes coding HCV antigens were cloned and sequenced, chimeric polyproteins containing four immunodominant components (core, NS3, NS4 and NS5) were expressed in E. coli both in soluble and in inclusion body form. These two chimeric proteins retained the antigenicity of HCV antigens. Antibody-capturing ELISA using the chimeric antigens showed a sensitivity of 97% (97/100) and a specificity of 98% (97/99) using the reference panel from the National Institute for the Control of Pharmaceutic and Biological Products of China (NICPBC); the same assay showed a sensitivity of 97.9% (48/49) and a specificity of 100% (43/43) using the self-established reference panel. Antigen-capturing ELISA was set up using the antigen labeled with horseradish peroxidase as conjugate, and was shown to be as sensitive as (97.9%) and more specific than (100%) antibody-capturing ELISA using the reference panel in this work. The antigen-capturing ELISA also showed a high accordance (98.9%) with UBI HCV enzyme immunoassay (EIA) 4.0 kits (United Biomedical Inc. USA). CONCLUSION: Antigen-capturing ELISA provided a convenient, sensitive and more specific approach for the diagnosis of hepatitis C virus infection.
BACKGROUND:Hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis. Detection of circulating antibodies against HCV by enzyme-linked immunosorbent assay (ELISA) has provided the main approach for the diagnosis of HCV infection. Most ELISA kits use a mixture of core, NS3, NS4 and NS5 antigen as capture antigens and enzyme-labeled goat anti-human IgG as conjugate. OBJECTIVES: To establish an ELISA system based on the antigen-capturing principle, using a recombinant chimeric polyprotein containing four HCV antigenic components as antigen. STUDY DESIGN:HCV antigens were expressed in Escherichia coli as chimeric polyprotein either in inclusion bodies or in soluble form. Protein expressed in inclusion bodies was used as solid-phase antigen, and the antigen expressed in a soluble form was used as enzyme conjugate after being labeled with horseradish peroxidase (HRP). RESULTS: Genes coding HCV antigens were cloned and sequenced, chimeric polyproteins containing four immunodominant components (core, NS3, NS4 and NS5) were expressed in E. coli both in soluble and in inclusion body form. These two chimeric proteins retained the antigenicity of HCV antigens. Antibody-capturing ELISA using the chimeric antigens showed a sensitivity of 97% (97/100) and a specificity of 98% (97/99) using the reference panel from the National Institute for the Control of Pharmaceutic and Biological Products of China (NICPBC); the same assay showed a sensitivity of 97.9% (48/49) and a specificity of 100% (43/43) using the self-established reference panel. Antigen-capturing ELISA was set up using the antigen labeled with horseradish peroxidase as conjugate, and was shown to be as sensitive as (97.9%) and more specific than (100%) antibody-capturing ELISA using the reference panel in this work. The antigen-capturing ELISA also showed a high accordance (98.9%) with UBI HCV enzyme immunoassay (EIA) 4.0 kits (United Biomedical Inc. USA). CONCLUSION: Antigen-capturing ELISA provided a convenient, sensitive and more specific approach for the diagnosis of hepatitis C virus infection.